The present invention relates generally to the use of lipocalin 2 as a biomarker for IL-17 mediated diseases and for monitoring the response of a patient to anti-IL-17 therapy.
Interleukin 17 (IL-17), also known as CTLA-8 or IL-17A, is a pro-inflammatory cytokine which stimulates the secretion of a wide range of other cytokines from various non-immune cells. IL-17 is capable of inducing the secretion of IL-6, IL-8, PGE2, MCP-1 and G-CSF by adherent cells like fibroblasts, keratinocytes, epithelial and endothelial cells and is also able to induce ICAM-1 surface expression, proliferation of T cells, and growth and differentiation of CD34+ human progenitors into neutrophils when cocultured in presence of irradiated fibroblasts (Fossiez et al., 1998, Int. Rev. Immunol. 16, 541-551). IL-17 is predominantly produced by activated memory T cells and acts by binding to a ubiquitously distributed cell surface receptor (IL-17R) (Yao et al., 1997, Cytokine, 9, 794-800). It may also act through binding to a complex of IL-17RA and IL-17RC (Toy et al., 2006, J. Immunol. 177(11); 36-39). A number of homologues of IL-17 have been identified which have both similar and distinct roles in regulating inflammatory responses. For a review of IL-17 cytokine/receptor families see Dumont, 2003, Expert Opin. Ther. Patents, 13, 287-303.
The most closely related homologue is IL-17F (ML-1), which shares approximately 55% amino acid sequence homology with IL-17A (Moseley et al., 2003, Cytokine Growth Factor Rev. 14: 155-174). IL-17A and IL-17F are expressed by the recently defined autoimmune related subset of T helper cells, Th17, that also express IL-21 and IL-22 signature cytokines (Korn et al., 2009, Annu. Rev. Immunol. 27:485-517.: 485-517). IL-17A and IL-17F are expressed as homodimers, but may also be expressed as the IL-17A/F heterodimer (Wright et al. 2008, J. Immunol. 181: 2799-2805). IL-17A and F signal through the receptors IL-17R, IL-17RC or an IL-17RA/RC receptor complex (Gaffen 2008, Cytokine. 43: 402-407). Both IL-17A and IL-17F have been associated with a number of autoimmune diseases.
IL-17 may contribute to a number of diseases mediated by abnormal immune responses, such as rheumatoid arthritis, multiple sclerosis and air-way inflammation, as well as organ transplant rejection and antitumour immunity. Inhibitors of IL-17 activity are well known in the art, for example an IL-17R:Fc fusion protein was used to demonstrate the role of IL-17 in collagen-induced arthritis (Lubberts et al., J. Immunol. 2001, 167, 1004-1013), neutralising polyclonal antibodies have been used to reduce peritoneal adhesion formation (Chung et al., 2002, J. Exp. Med., 195, 1471-1478) and a neutralising antibody to IL-17 was used to demonstrate the role of IL-17 in Experimental Autoimmune Encephalomyelitis, a murine model of MS (WO2005/051422). Numerous neutralising anti-IL-17A antibodies have been described, see for example those described in WO2006/054059, WO2006/013107, WO2007070750 and WO2007149032. Antibodies which bind both IL-17A and IL-17F have also been described, see for example, WO2007/106769, WO2008/047134, WO2009/136286 and WO2010/025400.
The role of biomarkers is becoming increasingly important in the clinical development of therapeutics. A biomarker can be an indicator of normal biological processes, disease processes or pharmacological responses to therapeutic intervention. Their role ranges from stratifying the patient population in helping to identify responders versus non-responders, to determining the dosing and efficacy of the therapeutic. Accordingly, biomarkers can be valuable tools in making better decisions that will reduce the cost for drug development and target therapies to the most suitable patient population.
Lipocalins are small secreted proteins with a common tertiary structure that binds lipophilic molecules such as prostaglandins and cholesterol and some are known to be biomarkers of certain diseases (Xu and Venge, 2000, Biochimica et Biophysica Acta, 1482, 298-307). One such lipocalin is lipocalin 2 (LCN2), also known as 24p3 or neutrophil gelatinase-associated lipocalin (NGAL) which is a 25 kDa secretory glycoprotein that was originally identified in mouse kidney cells and human neutrophil granules. LCN2 has been demonstrated to be a biomarker for early detection of various renal injuries, obesity and cancer (see for example, Devarajan, 2007, Contributions to Nephrology, 156, 203-212; Wang et al., 2007, Clinical Chemistry, 53, 34-41; Ratana et al, 2007, International Journal of Cancer, 120 (11), 2426-34). LCN2 gene expression has been shown to be upregulated in Experimental Autoimmune Encephalomyelitis, a murine model of MS (Jelinsky et al., 2005, Journal of Neurological Sciences, 239, 81-93). IL-17 has been demonstrated to be involved in the transcriptional regulation of LCN2 in vitro via NF-κB and C/EBP (Shen et al., 2006, Journal of Biological Chemistry, 281, 34, 24138-24148; Shen et al., 2005, Journal of Leukocyte Biology, 77, 388-399). Whether IL-17 regulates LCN2 in vivo is not known.
The present invention demonstrates that LCN2 can serve as a biomarker for IL-17 mediated diseases and for anti-IL-17 therapy. Specifically we have been able to demonstrate that a neutralising anti-IL-17 antibody reduces LCN2 expression in an animal model of MS. Hence, in one embodiment the present invention provides a method of monitoring the response of a subject to a medicament comprising an IL-17 inhibitor, wherein the level of expression of lipocalin 2 in a test sample of a body fluid or tissue obtained from the subject is assessed before and after administration of said medicament and wherein the level of expression of lipocalin 2 after administration of the medicament is compared to the level of expression of lipocalin 2 prior to administration of the medicament. The levels of expression of lipocalin 2 in the subject before and after administration of the medicament are compared in order to determine the efficacy of the IL-17 inhibitor therapy.
In the present application the use of the term ‘IL-17’, as described herein above, refers to the protein also known as CTLA-8 or IL-17A.
In the method of the present invention the level of expression of LCN2 may be measured in any suitable test sample of a body fluid or tissue obtained from the subject. Suitable test samples include, but are not limited to, blood, serum, plasma, urine, tissue biopsy, stool, sputum, cerebrospinal fluid and bronchioalveolar lavage (BAL) fluid.
Suitable methods for determining the level of expression of LCN2 in a test sample are known in the art and these include, but are not limited to, immunoassays, gel electrophoresis followed by visualisation, detection of mRNA or measurement of LCN2 activity (e.g. iron shuttling, see for example Schmidt-Ott et al., J. Am. Soc. Nephrol. 2007, 18 (2) 407-413).
In one embodiment the level of expression of LCN2 is determined by measuring the level of LCN2 polypeptide in a test sample.
In one embodiment, the step of detecting the expression level of the LCN2 polypeptide comprises:                (a) contacting the test sample with a capture reagent that is specific for a LCN2 polypeptide; and        (b) detecting whether binding has occurred between the capture reagent and said LCN2 polypeptide in the sample.        
In one aspect, the captured LCN2 polypeptide is detected using a directly or indirectly labelled detection reagent which may be immobilised on a solid phase.
A convenient means for detecting/quantifying a LCN2 polypeptide involves the use of antibodies. The LCN2 polypeptide can be detected by means of any immunoassay known in the art, including, without limitation, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, 2 dimensional gel electrophoresis, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays.
Detection of the interaction of an antibody with LCN2 can be facilitated by coupling the antibody to a detectable substance for example, but without limitation, an enzyme (such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, acetylcholinesterase), a prosthetic group (such as streptavidin, avidin, biotin), a fluorescent material (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin), a luminescent material (such as luminol), a bioluminescent material (such as luciferase, luciferin, aequorin), a radioactive nuclide (such as 125I, 131I, 111In, 99Tc) a positron emitting metal or a non-radioactive paramagnetic metal ion (see U.S. Pat. No. 4,741,900).
Examples of suitable assays for the detection of LCN2 are described by Xu et al., 1994, Journal of Immunological Methods, 171, 2, 245-252 and Flo et al., 2004, Nature 432, 917-921.
It will also be apparent to one skilled in the art that the level of expression of LCN2 may be determined by measuring the level of an LCN2 nucleic acid, preferably mRNA.
Nucleic acid detection may be achieved using any suitable method known in the art including for example, hybridisation assays, arrays of capture probes (mRNA or cDNA), amplification e.g. PCR, RT-PCR (see for example the methods described in WO2006/125105).
In the present invention the response of a subject to a medicament comprising an IL-17 inhibitor is monitored by measuring the level of expression of LCN2 in test samples obtained from the test subject before and after administration of said medicament. Preferably the level of expression of LCN2 is measured in samples taken over successive time intervals following administration of the medicament. For example LCN2 expression can be determined one or more times following administration of the medicament, e.g. over the course of therapy e.g. over the course of one, two, four, six weeks, six months or longer after initiating a therapeutic regimen.